A monoclonal antibody is prepared by fusing antibody producing cells of an experimental animal (usually, a mouse) and myeloma cells to prepare hybridomas and screening the hybridomas to select a strain producing an intended antibody. Georges J. F. Kohler and Cesar Milstein who invented a preparation method of a monoclonal antibody in 1975 won the Nobel Prize in Physiology or Medicine in 1984. For screening of hybridomas that secrete an intended monoclonal antibody, ELISA is typically employed and wells providing high activity are subjected to single cell cloning. As a result, fusion cells in the wells that provide high activity are hybridomas producing the intended monoclonal antibody.
Antibodies produced by the hybridomas obtained by single cell cloning however often fail to reproduce the activity obtained in first ELISA. There are presumed to be two reasons. First one is that hybridomas that are producing an intended antibody stop their growth when they are placed in one well (of, for example, a 96-well plate) and a clone cannot be established. The second one is that the although a clone can be obtained, an intended antibody gene is lost during subcloning from a 96-well plate to a 24-well plate, a 6-well plate, and a 10-cm dish, making it difficult to establish them as hybridomas.
The reason why the activity is not reproduced in single cell cloning has not yet been elucidated at all, but is presumed that an antibody gene intrinsic to myeloma cells may exclude an intended antibody gene introduced later by cell fusion. There is also a possibility that either an antibody gene derived from myeloma cells or an intended antibody gene derived from antibody producing cells is excluded stochastically. Anyway, the intended antibody gene tends to be excluded because it is unnecessary for the survival of hybridomas.